1. Field of the Invention
The invention relates to the field of electrochemical detectors for capillary electrophoresis, and more particularly to a method for fabricating such detectors and the resulting structure realized from the fabrication techniques.
2. Description of the Prior Art
In simplified terms, capillary zone electrophoresis is the separation of a chemical material along the length of a capillary column filled with a buffered or neutral solution. By the application of a high DC voltage, typically in the order of 30,000 volts, materials in an unknown sample are driven from the positive anode toward a negative anode at the detector end of the column at varying rates. As these charged constituents reach the detector near or at the grounded anode of the electrophoresis column, small signals are detected. The time of arrival of the constituent is indicative of the type of material and is used for qualitative analysis. The amplitude of the detected signal can be calibrated against standardized samples to measure the amount of material or its concentration within the sample.
The particular advantage of capillary zone electrophoresis is the very small amount of sample that is needed in order to obtain an electrochemical signal. Typically, the samples measure in the range of nanoliters and practitioners in the art have achieved capillary electrophoresis with samples of the order of picoliters, which approaches the size of many constituents within a single living cell.
Capillary zone electrophoresis is a powerful separation technique which is ideally suited for the analysis of charged species or materials. See J. W. Jorgenson, ACS Symposium Series 335, pages 182-198 (1987). One of the major limitations with capillary zone electrophoresis, however, is related to detection in the eluted zone. See J. W. Jorgenson et al., Science, 222, pages 266-72 (1983). Since small diameter capillaries, typically with an inner diameter of less than 75 microns, are required for efficient separation, detection of the sample must be made in the capillary column itself. Many different types of detection strategies have been implemented in capillary zone electrophoresis, see for example the review of R. Wallingford et al., Advances in Chromatography Series, 29, pp 1-76 (1989). The most sensitive techniques found to date involve laser induced fluorescence, see P. Gozel, Anal. Chem., 59, pages 44-9 (1987); D. E. Burton et al., Chromatogr. Sci. 24, pages 347-51 (1986); and W. G. Kuhr et al., Anal. Chem. 60, pp 1832-4 (1988) and involve electrochemical detections, see R. A. Wallingford et al., Anal. Chem. 60, pages 258-63 (1988); and R. A. Wallingford et al., Anal. Chem. 59, 678-81 (1987). Attomole detection limits, i.e. 10.sup.-18 injected mole of sample, have been demonstrated with these techniques, but only a few chemical species have sufficient fluorescent efficiency or electrochemical activity to enable full utilization of these types of detectors. See W. G. Kuhr, Anal. Chem. 62, pp R403-14 (1988); and R. Wallingford et al., Anal. Chem. 60, 1972-75 (1988).
Electrochemical detection has been shown to have virtually the same level of sensitivity when used with capillary zone electrophoresis, but electrochemical detection is much less expensive to perform and has more general applications. Extremely sensitive electrochemical measurements can be made using capillary zone electrophoresis by electrically decoupling an amperometric detector from the electrophoretic power supply. See R. Wallingford et al., Anal. Chem., 59, 1962-66 (1987). According to this art, a small break in the separation capillary several centimeters before the detector acts as an electrically conductive joint to remove current from the separation column without disturbing the flow of the fluid buffer along the capillary column. In this technique, a carbon fiber typically with a radius of 5 to 10 microns and 100 to 500 microns in length is inserted into the end of the capillary column with the reference electrode placed in the buffer solution at the end of the capillary. By this combination, an amperometric detector with a volume of only of the order of a few tens of picoliters is provided. Careful matching of the outer diameter of the carbon electrode fiber with the inner diameter of the capillary allows nanomolar detection limits to be easily obtained for indoles, catecholamines and their metabolites.
One of the major problems with a detector as just described arises in the fabrication of the capillary tube relative to the carbon fiber electrode. A very small electrical joint with a width of less the 1 micron must be introduced into the separation column and the two segments of the capillary must remain perfectly aligned to allow the flow of the analyte to continue uninterruptedly along the capillary column to the detector. In addition, insertion of the carbon fiber into the separation capillary is performed using micromanipulators which are expensive and delicate mechanical devices requiring considerable talent and finesse for their successful operation. Further, electrophoretic columns using micromanipulators have a design such that their use with vacuum injection systems, which can be very helpful in filling and purging the capillary column, is very difficult if not impossible.
Therefore, what is needed is some type of capillary zone electrophoresis which can be deployed in the separation column and achieve high levels of sensitivity of electrochemical detection without the need or difficulty of using micromanipulators, and further which is rugged in its design such that critical misalignment of the capillary in the fiber cannot occur under normal handling and use.